Field of the Invention
Embodiments of the present invention relate in general to methods and compositions for amplifying genomic sequences, such as the whole genome of a single cell.
Description of Related Art
Formation of primer dimers is a common problem in existing methods for DNA or RNA amplification using random primers. In order to achieve efficient priming for each individual sequence, random primers must be applied at very high concentrations. The efficiency of annealing to a specific target DNA or RNA template or the entire population of template molecules is greatly reduced by the formation of primer-dimers resulting from the high primer concentrations required for efficient priming. Other problems known in the art when using random primers to amplify DNA include an inability to amplify the genome in its entirety due to locus dropout (loss), generation of short amplification products, and in some cases, the inability to amplify degraded or artificially fragmented DNA.
Methods directed to aspects of whole genome amplification are reported in the literature. See, for example, U.S. Pat. No. 7,718,403, US 2003/0108870 and U.S. Pat. No. 7,402,386. PCR-based methods such as GenomePlex and Picoplex can induce significant amplification bias across different loci in the genome which results in significant amplification loci dropout. With these methods, only 5-30% of the genome is often sequenced with reasonable sequencing depth from amplified single cells. Multiple displacement amplification utilizes low temperature amplification which leads to large amounts of chimera sequences that do not belong to the original genome resulting in significant false discovery rate of one out of several kilobases and introduces artifacts into the whole genome sequencing analysis. While methods for whole genome amplification have been proposed, such methods can generally be inefficient, complex and expensive. Therefore, a need exists for alternate methods of amplifying small amounts of genomic DNA, such as from a single cell or small group of cells.